INVESTIGADORES
IBAÑEZ Lorena Itati
congresos y reuniones científicas
Título:
PRELIMINARY STUDIES FOR THE EXPRESSION AND PURIFICATION OF TWO PROTEINS FROM Candidatus Liberibacter asiaticus
Autor/es:
SPERAT W; BIANCO I; TORRES P; PAGLIALI F; GONZALEZ C; IBAÑEZ LI; VOJNOV A
Lugar:
Buenos Aires
Reunión:
Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017
Institución organizadora:
SAIC, SAIB, SAI, SAA, SAB, SAFE, SAFIS, SAH, SAP
Resumen:
Huanglongbing (HLB), disease caused by bacteria of the genus Candidatus Liberibacter (CaLib), is having devastating effects on the citrus industry worldwide. However, despite its economic importance, little is known about the biology and pathogenesis mechanisms of these bacteria. The main hurdle in the study of CaLib is that only one specie is culturable in vitro, Candidatus Liberibacter crescens, which is not described as pathogen. Of all the HLB-causing species, Candidatus Liberibacter asiaticus (CaLas) is the most widespread and studied. Several genomes of CaLas have been sequenced.By analyzing both the sequenced genomes and the recent publications on the subject, we have selected two proteins that could potentially be involved in the pathogenesis of this bacterium: CLIBASIA_04040 (4040), an uncharacterized putative protein, and CLIBASIA_04260, the CaLas OmpA homologue (OmpA). We amplified the sequences corresponding to the 4040 protein (without the N-terminal signal peptide) and the N-terminal soluble domain of OmpA. Both open reading frames were cloned into the expression vector pET28a(+). Then, we overexpressed 4040 and OmpA in Escherichia coli BL21(DE3) and E. coli Rosetta-gami 2, respectively, both with a hexahistidine-coding sequence. Using differential centrifugation, we detected that the 4040 protein was localized in the soluble fraction (cytoplasm or periplasm), while OmpA was localized in the insoluble fraction (inclusion bodies). OmpA was solubilized by heating the insoluble fraction at to 60 °C for 20 minutes. Our next step is to purify these proteins by affinity chromatography to analyze them in vitro.